In the course of studying inflammatory muscle diseases (polymyositis, dermatomyositis, and related diseases), we have encountered patients with other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid "-glucosidase, or GAA) deficiency. Acid maltase deficiency is fatal in childhood if the enzyme is absent and leads to a progressive proximal myopathy with pulmonary failure secondary to diaphragmatic involvement in later decades if some enzyme is present. Earlier studies in our laboratory of the most common adult mutation with an in vitro model system have shown that a single base mutation in the polypyrimidine tract towards the end of intron 1 reduces the transcription rate, apparently by altering the binding of a splicing factor, and alters the ratio of splice variants to favor the splicing of non-productive mRNA. Furthermore, a silencer was identified elsewhere in this intron, and was considered a possible target for therapeutic intervention. If the silencer could be overcome, the resulting upregulation of gene transcription might overcome the reduced synthesis in the common adult allele. In the past year, we have tested and rejected for further trials a number of compounds that non-specifically upregulate some genes (hydroxyurea, retinoic acid, butyrate). Early attempts to identify conditions that upregulate all lysosomal enzymes have also been unsuccessful. Attention is now therefore focused on pinpointing the silencer activity and finding the involved transcription factors. The region has been localized to a 152 bp stretch of IVS1, and gel shift of this region is induced by nuclear extracts. Within this region are binding motifs for several known transcription factors. These studies continue actively. In order to provide an animal model for testing several proposed therapies for acid maltase deficiency which are actively underdevelopment in our group and by groups in the Netherlands, in New York, at Duke, and at Johns Hopkins, we have made knockout models of the GAA gene in mice. With the assistance of Dr. Brian Sauer of NIDDK and Dr. Edward Ginns of NIMH, we have created complete knockouts of the gene by the introduction of the neo gene in exon 6 and in exon 14. Homozygous F2 offspring rapidly accumulate glycogen in cardiac and skeletal muscle. Females show impaired performance in muscle testing by quantitative open field observation and by ability to hang on a wire screen or move on a rotating rod, and by several months, they develop a grossly waddling gait. The exon 6 knockout was designed so that the neo gene and the whole of exon 6 could be deleted permanently by mating the mice to mice transgenic for the CRE recombinase. These mice delta 6/delta 6 have a slower onset of clinical disease despite total absence of GAA activity, apparently because of differences in the background genes of the two strains. The details of these experiments have been published recently, and the mice have been distributed to groups around the world which are working on therapy by enzyme or by gene replacement. We are attempting to develop a mouse disease model with a more rapid course by breeding the mice to mice which are transgenic either for glycogen synthetase or for the glucose transporter, GLUT1. Our own efforts at gene therapy began with in vitro studies of a promising retroviral vector which were published last year. We are currently attempting to develop a better retroviral vector for treatment of the knockout mice. In addition, we have begun studies of therapeutic bone marrow transplantation. The members of the group have joined with others studying acid maltase deficiency to organize an international workshop on the disease to be held at NIH in December 1998. We have extended studies of a mutation in the GAA gene in African and African-American patients with acid maltase deficiency. We have established the probable tribal origin of the mutation. Studies of the intragenic polymorphisms clearly establish that all the examples of this mutation (R854X) in Africans and African-Americans have a common origin, thus allowing the inference that the African-Americans share common ancestors with the Africans. The success of this modest project - carried out with the help of scientific collaborators from around the world and with the close collaboration of John Vlach, Professor of Anthropology at George Washington University - shows the way in which genetic information can be used to help African-Americans gain much more precise knowledge of their ancestry. The results of this study were recently published and were presented in a Clinical Center Videoconference Grand Rounds.